Decreased T lymphocyte functional responses are consistently found in advanced age in humans. Advanced age is also associated with a decrease in antigenically "naive" or "virgin" T cells and an increase in numbers of "memory" T cells that mediate recall responses in human peripheral blood. The specific focus of this project is to determine the functional linkage between this change in peripheral blood T cell subsets and the diminished activation responses of peripheral human T cells observed in vitro in advanced age. This proposal rests on the hypothesis that persistence of memory T cells leads to increased average biological age of these cells in old subjects, compared to young donors. The increased age of this subset results in T cells that become refractory to activation stimuli leading to the decline in in vitro activation responses, with little or no effect on the functional responses of the "naive" T cell subset. The Specific Aims of this proposal are: 1) To determine the effects of advanced age on surface marker expression and physical properties of human "naive" and "memory" T cells. The experimental approach for this Aim will be to characterize T cell subsets from older subjects by determining expression of various adhesion receptors on the cell surface, distribution of cell size, density and RNA/DNA content, membrane microviscosity and lateral diffusion of membrane proteins. 2) To determine the effects of advanced age on functional responses of "naive" and "memory" T cell subsets. Purified populations of "naive" and "memory" T cells will be analyzed by a variety of functional assays including proliferative responses to mitogens and recall antigens, lymphokine secretion, receptor expression following activation, increase of intracellular free calcium by both bulk culture and single cell assays, and the kinetics of conversion of naive to memory cells following activation. 3) To quantitate, isolate and characterize T cells that become refractory to activation stimuli in advanced age. We will use the uptake of merocyanine dye as an early marker of activation, expression of the IL-2 receptor, the protooncogene products c-myc and c-myb and DNA content to quantitate the numbers of responsive and nonresponsive cells. These methods will allow surface marker characterization and sorting of viable cells. The nonresponding cells will be examined for membrane alterations, heat shock response, proliferative responses to PMA and ionomycin and changes in nuclear function. The results are expected to lead to insights not only into the basis of depressed immune function in advanced age, but also into the basic mechanisms of in vivo cellular aging.